Aneuploidy is a common form of chromosome mutation wherein the number of individual chromosomes present in a cell increases or decreases from the number present in a normal cell. The absence of one chromosome from the diploid complement is referred to as monosomy, whereas the presence of an extra chromosome is referred to as trisomy. Trisomy 21 is a condition where an extra chromosome 21 exists and is the most common form of aneuploidy that gives rise to Down's syndrome, a congenital manifestation of potentially severe mental retardation.
Prenatal diagnosis of chromosomal disorders using karyotypes in combination with amniotic fluid or chorionic villus sampling has become the standard in highly developed countries worldwide in instances where indications are present. A karyotype is a collection of indices which characterize the state of a chromosomal complement; it includes total chromosome number, copy number of individual chromosome type and chromosome morphology. Karyotypes are determined by staining metaphase (or condensed) chromosomes. Metaphase chromosomes were used often because, until recently, it had not been possible to visualize interphase chromosomes because of their dispersed condition and the lack of sensitivity from such stainings. Metaphase chromosomes can be stained using a number of cytological techniques and reveal a longitudinal segmentation into entities generally referred to as bands. The banding pattern of each chromosome is unique and thus permits unambiguous chromosome identification including any abnormalities. Unfortunately, such analysis requires cell culturing and the preparation of high quality metaphase chromosomes, which may take 1-3 weeks.
Some attempts have been made to analyze chromosomes without cell culturing, and one such method is described in U.S. Pat. No. 5,447,841. A rapid partial karyotype analysis is obtained using fluorescence in situ hybridization (FISH) with respect to the chromosome-specific DNA targets which are present upon uncultured interphase amniotic cells. In this approach, fluorescently labeled probes are hybridized to complementary sites on chromosomes bound to a solid support. This approach overcomes a limitation of conventional cytological staining methods which results from a lack of stains that are sufficiently chromosome-specific; reagents comprising heterogeneous mixtures of labeled nucleic acid fragments are provided that can be hybridized to the DNA of specific chromosomes, specific subsets of chromosomes or specific subregions of specific chromosomes. Although the FISH method opens up the possibility of rapid and highly sensitive detection of chromosomal disorders in both metaphase and interphase cells, it is not without some shortcomings: 1) a number of chromosomal disorders related to microdeletions require still metaphase chromosomes due to the lack of their signal sensitivity and 2) the number of chromosomes which can be simultaneously diagnosed is limited to the resolution of the fluorescence microscope filters. Moreover, performance of the FISH method requires highly trained technicians and expensive equipment and reagents.
A method for quantitating cDNA species by PCR by coamplifying a second, unrelated template has been described, see Rappolee, D. A., et al., Science 241: 708-712 (1988). This method is critically dependent on certain variables, including cycle number and amount of starting mRNA of each species. Even when these variables are adequately controlled, it is unlikely that an unrelated control template will be amplified at precisely the same rate as the unknown template. Small differences in the rate of amplification of the two templates are magnified during PCR and may grossly over- or underestimate the amount of the unknown template present in the ultimate assay that is used.
It was reported in Amhelm, Norman et al. Polymerase Chain Reaction, Chemical & Engineering News, 36-47 (Oct. 1, 1990), that PCR has been used for the prenatal diagnosis of genetic diseases to analyze fetal DNA to determine whether it contains a mutated form of a gene that would cause the disease. Following PCR amplification, labeled probes are used to test a PCR sample for the presence of the disease causing allele. It is stated that the presence or absence of several different causing genes can be determined in a single sample. It is further indicated that PCR detection methods have employed reporters, based upon enzymes, chemiluminescence and fluorescence, in detection methods to find a pathogen using a dot blot format by spotting PCR-amplified product on a nylon membrane and then exposing that dot-carrying membrane to a single stranded DNA probe that will specifically anneal to the PCR product of interest, the probe being labeled radioactively or with molecules that are detectable by fluorescence or chemiluminescence. This procedure was limited to screening for one analyte at a time. Alternatively described is the use of PCR to label the pathogen-specific product itself by incorporating a label in the side chain of the nucleotides where it will not interfere with the ability of the primer to anneal to the target or to be extended by the polymerase. The incorporation of fluorescent dyes, such as fluorescein, or the vitamin biotin are mentioned, that might be attached by a short linker arm to the 5′ end of the primer or to one of the bases within the primer sequence. It is then suggested that, once the appropriately labeled, amplified PCR product has been produced, it can be detected by “capturing” with a non-labeled pathogen-specific probe attached to a solid support. This alternative process was referred to as the reverse dot blot procedure when it was developed at Cetus Corporation. After washing to get rid of all excess labeled-primers or the like, the presence of the analyte is detected as by binding to the bacterial protein streptavidin and then treating with an enzyme. Another capture suggestion is to label one primer with a fluorescent dye and the other with biotin and then capture the double-stranded product with streptavidin and test for the fluorescence. It is mentioned that this capture approach will allow the simultaneous detection of different diagnostic sequences by using sets of primers for different specific pathogens one of which is labeled. It is suggested that the total PCR product is hybridized to a suitable nylon membrane support having distinct regions, each with a capture probe that is specific for the PCR product of one of the pathogens. Then, the support is stringently washed following hybridizing and if the label was biotin, the addition of the strepavidin-enzyme complex followed by the appropriate enzyme substrate would indicate the presence of any one of the pathogens by color production in that specific region. It is suggested that this procedure might be preferred over the original dot blot procedure which would involve many more manipulations because it would require that a different aliquot of the unlabeled PCR product be tested with each labeled, pathogen-specific probe in a separate hybridization experiment.
U.S. Pat. No. 5,213,961 to Bunn et al. discloses a method of quantitative PCR by competitive methodology, wherein the parameters affecting DNA amplification and a mechanism to distinguish differences in template (both test and control) ratios and copy numbers are discussed. It is mentioned that it might be used to detect somatic cell mutations. Bunn et al. address the effect of various parameters on the amplification process which arise predominantly from the nature of the DNA primers and their respective primer binding sites; however, the system is limited to use of a standard that is sufficiently close to the target that the target and sample are co-amplified at the same rate by PCR. Moreover, the standard must differ from the target such that its length can be later altered by enzymatic action, thus allowing the standard and target to be separated and quantified by electrophoresis.
PCT published application WO 94/03638 discloses a different method where aneuploidy is detected by utilization of short tandem repeat DNA sequences present in chromosome DNA, with PCR methodology being utilized to amplify the short tandem repeat sequences.
U.S. Pat. No. 5,888,740 to Han uses DNA templates engineered as internal controls during PCR reactions for the quantitative measurement of genomic DNA levels. These control DNA templates are designed to have the same sequences of primers for PCR reaction as the test sample DNA templates. The goal is for amplification to proceed at the same rate, thereby providing control of the amplification rate. However, this method requires a specific internal control for each chromosome, which is complex. In addition, the PCR reaction for each chromosome must be carried out in a separate well of a microtiter plate or other container.
U.S. Pat. No. 6,551,783 to Carey teaches a method of quantitation of expression of two target genes in a simultaneous PCR based assay which employs a system where a fluorescent marker or reporter probe is displaced from the strand of DNA being amplified in a manner in which the fluorescent probe escapes from the vicinity of a quencher dye. By using two different fluorescent probes, PCR multiplexing of two genes are simultaneously effected. One target gene is a ubiquitously expressed marker gene, such as GAPDH or DAD-1. The method taught is limited to one unknown target at a time.
Another method, described in WO94/23023, quantifies a target gene based on multiplex PCR for the target gene, a housekeeping gene and their competitive templates with mutations, such as point mutations, insertions or deletions. This method is complex and cumbersome, employing gel electrophoresis in the final analysis.
None of the aforementioned methods of screening for chromosomal disorders is felt to be totally satisfactory to screen for multiple disorders simultaneously, so the search has continued for a comprehensive screening procedure that is easy to use and provides rapid results.